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Distinct classes of potassium channels fused to GPCRs as electrical signaling biosensors.

ABSTRACT: Ligand-gated ion channels (LGICs) are natural biosensors generating electrical signals in response to the binding of specific ligands. Creating de novo LGICs for biosensing applications is technically challenging. We have previously designed modified LGICs by linking G protein-coupled receptors (GPCRs) to the Kir6.2 channel. In this article, we extrapolate these design concepts to other channels with different structures and oligomeric states, namely a tetrameric viral Kcv channel and the dimeric mouse TREK-1 channel. After precise engineering of the linker regions, the two ion channels were successfully regulated by a GPCR fused to their N-terminal domain. Two-electrode voltage-clamp recordings showed that Kcv and mTREK-1 fusions were inhibited and activated by GPCR agonists, respectively, and antagonists abolished both effects. Thus, dissimilar ion channels can be allosterically regulated through their N-terminal domains, suggesting that this is a generalizable approach for ion channel engineering.

SUBMITTER: Garcia-Fernandez MD

PROVIDER: S-EPMC8688152 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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Distinct classes of potassium channels fused to GPCRs as electrical signaling biosensors.

García-Fernández M Dolores MD Chatelain Franck C FC Nury Hugues H Moroni Anna A Moreau Christophe J CJ

Cell reports methods 20211220 8

Ligand-gated ion channels (LGICs) are natural biosensors generating electrical signals in response to the binding of specific ligands. Creating <i>de novo</i> LGICs for biosensing applications is technically challenging. We have previously designed modified LGICs by linking G protein-coupled receptors (GPCRs) to the Kir6.2 channel. In this article, we extrapolate these design concepts to other channels with different structures and oligomeric states, namely a tetrameric viral Kcv channel and the ...[more]

PMID: 34977850

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Project description:Voltage-gated K+ channels play central roles in human physiology, both in health and disease. A repertoire of inhibitors that are both potent and specific would therefore be of great value, not only as pharmacological agents but also as research tools. The small molecule RY785 has been described as particularly promising in this regard, as it selectively inhibits channels in the Kv2 subfamily with high potency. Kv2 channels are expressed in multiple cell types in humans, and are of particular importance for neuronal function. The mechanism of action of RY785 has not yet been determined at the molecular level, but functional studies indicate it differs from that of less specific inhibitors, such as quaternary-ammonium compounds or aminopyridines; RY785 is distinct also in that it is electroneutral. To examine this mechanism at the single-molecule level, we have carried out a series of all-atom molecular dynamics simulations based on the experimental structure of the Kv2.1 channel in the activated, open state. First, we report a 25-microsecond trajectory calculated in the absence of any inhibitor, under an applied voltage of 100 mV, which demonstrates outward K+ flow under simulation conditions at rates comparable to experimental measurements. Additional simulations in which either RY785 or tetraethylammonium (TEA) is introduced in solution show both inhibitors spontaneously enter the channel through the cytoplasmic gate, with distinct effects. In agreement with prior structural studies, we observe that TEA binds to a site adjacent to the selectivity filter, on the pore axis, thereby blocking the flow of K+ ions. RY785, by contrast, binds to the channel walls, off-axis, and allows K+ flow while the cytoplasmic gate remains open. The observed mode of RY785 binding, however, indicates that its mechanism of action is to stabilize and occlude a semi-open state of the gate, by bridging hydrophobic protein-protein interactions therein; this hypothesis would explain the puzzling experimental observation that RY785 recognition influences the gating currents generated by the voltage sensors, 3 nm away.

Dataset Information

Distinct classes of potassium channels fused to GPCRs as electrical signaling biosensors.

Publications

Distinct classes of potassium channels fused to GPCRs as electrical signaling biosensors.

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